The present invention relates to synthetic peptides, the sequences of which correspond to antigenic regions of an immunologically important protein of HTLV-1. These peptides are useful as diagnostic reagents for detecting the presence of antibodies to HTLV-1 and may also be useful as immunogens in compositions and methods to elicit antibodies against HTLV-1 in animals and man.
The etiologic agent of adult T-cell leukemia/lymphoma (ATL) has been identified as HTLV-1 (human T-cell lymphotropic virus type 1). See e.g., Sarngadharan et al., in Virology (1985) B. N. Fields et al., eds., pp. 1345-1371, for a review. The region of the world where the virus is most prevalent is the island of Kyushu in southern Japan where about 15% of the population has been infected. Recently a tropical paralysis called tropical spastic paraparesis (TSP) has also been associated with HTLV-1 infection. Rodgers-Johnson et al., Lancet (1985) II:1247; Vernant et al., Ann. Neurol (1987) 21:123. In the tropics TSP is of the same magnitude and importance as the multiple sclerosis syndrome is in the western world. Marx, Science (1987) 236:1059-1061.
Methods for detection of HTLV-1 infection, in general, measure exposure to the virus by detecting and quantifying antibodies to HTLV-1 antigens in blood, sera, and blood-derived products. Such assays can be used to aid diagnosis of ATL and TSP and to screen blood and blood products for previous exposure to HTLV-1.
The current attempts to diagnose HTLV-1 infections and screen blood for exposure to HTLV-1 include enzyme-linked immunosorbent assay (ELISA) techniques to detect the presence of antibodies to immunogenic components of HTLV-1 in a test sample. Other methods may utilize Western blotting techniques to detect HTLV-1 specific antibodies in test samples. In general, almost any known immunoassay, such as radioimmunoassays, agglutination tests or indirect immunofluorescence, in addition to ELISA and Western blots, can be adapted, by use of specific reagents, for the detection of HTLV-1 and antibodies thereto.
The source of antigens for these assays may include inter alia antigenic proteins obtained from HTLV-1 infected T cell lines and antigens produced by recombinant DNA techniques. The use of antigens obtained from these sources, however, has significant drawbacks.
The production of HTLV-1 per se in continuous cell lines must be performed in high risk (P3 containment) laboratories due to the danger to investigators who may become adversely exposed to the virus. It is also likely that the use of T cell derived HTLV-1 antigens can produce false negative and false positive results in ELISA tests. For example, by analogy, in measuring exposure to the AIDS virus, there have been false negative and false positive results reported with ELISA tests using whole virus HIV-1 antigens obtained from cell lines. Gurtler et al., J Virological Methods (1987) 15:11-23. Western blot analyses for HTLV-1 detection, using electroblotted whole virus antigens, should provide greater specificity but are more laborious and time-consuming than ELISA tests. Furthermore, since HTLV-1 producing cells are of human origin, viral antigen preparations obtained from these cell lines, unless exhaustively purified, may be contaminated with normal cellular antigens, such as HLA antigens, which could produce false positive reactions in a ELISA test.
Exhaustive purification of viral antigens from cell lines can also conceivably destroy immunogenicity of immunologically important proteins or otherwise inactivate antigens, thereby producing reagents that result in false negative reactions. In addition, false negative reactions using live-virus-derived antigens may occur because of steric hindrance whereby antibodies cannot react with their specific antigens because the reaction is blocked by the presence of other antigens and antibodies in the reaction mixture.
ELISA tests to detect HTLV-1 infection may also employ immunologically important viral proteins produced by cloning portions of the HTLV-1 genome in bacteria. The complete nucleotide sequence of HTLV-1 has been reported by Seiki et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:3618-3622. The viral envelope glycoproteins and core proteins, respectively encoded by the env and gag genes of HTLV-1, are apparently the antigens recognized by antibodies in the sera of patients with HTLV-1 infection.
Immunologically important HTLV-1 antigens, which are present in the viral envelope and core, may be prepared by cloning portions of the HTLV-1 genome in various expression systems such as bacteria, yeast or vaccinia. Such recombinant antigens may be used in diagnosis and as potential vaccine compositions as has been done for HIV-1 proteins. See, e.g. Cabradilla et al., Biotechnology (1986) 4:128-133; Chang et al., Biotechnology (1985) 3:905-909; Putney et al., Science (1986) 234:1392-1395; Kieny et al. Biotechnology (1986) 4:790-795. HTLV-1 antigens produced by recombinant DNA methods, however, must still be exhaustively purified to avoid false positive reactions in the ELISA due to any antibody reactivity to antigens of the expression system which may contaminate the HTLV-1 antigen preparation. Also, denaturation of HTLV-1 antigens during purification may destroy important antigen activity.
While HTLV-1 antigens produced by recombinant techniques may be an improvement over antigens obtained from virus-infected cell cultures, the recombinant proteins still may not provide reagents that give as accurate a diagnosis as possible. Because of the nature of the disease and the need for accurate results, other reagents must be developed to approach 100% accuracy in diagnosis of HTLV-1.
Proteins contain a number of epitopes or antigenic determinants which are the regions of the proteins which comprise the binding sites for specific antibodies. In general, proteins contain between 5 to 10 epitopes, each of which comprises a sequence of 6 to 8 amino acids. Epitopes can be either continuous, in which the 6 to 8 amino acids are present in linear sequence, or discontinuous, in which case the amino acids that form the epitope are brought together by the three dimensional folding of the protein. Even though an epitope constitutes only a relatively few amino acids, its reactivity with an antibody is influenced by the amino acids in the protein which surround the epitope.
Studies aimed at mapping antigenic sites or epitopes of proteins have been aided by the use of synthetic peptides corresponding to various regions of the proteins of interest. See, e.g., Lerner et al., in The Biology of Immunological Disease: A Hospital Practice Book, (1983) Dixon and Fisher, eds., pp. 331-338; Lerner, Adv. Immunol. (1984) 36:1. In addition to their usefulness in epitope mapping studies, synthetic peptides, if encompassing major antigenic determinants of a protein, have potential as immunogenic compositions, including vaccines and diagnostic reagents. Synthetic peptide antigens have several advantages in specific antibody production and reactivity. The exact sequence of the peptide can be selected from the amino acid sequence as actually determined by amino acid sequencing of a protein or predicted from the DNA sequence coding for the protein. The use of specific synthetic peptides eliminates the need for using the full-length protein in the production of or assay for specific antibodies. Furthermore, the solid phase peptide synthetic techniques of Merrifield and coworkers allow for essentially unlimited quantities of the synthesized peptide of interest to be chemically produced. See, e.g., Erickson and Merrifield in The Proteins, 3rd Edit. (1976), Vol 2, Academic Press, New York, Chapter 3. The availability of automated peptide synthesizers has further advanced such techniques.
Although a variety of criteria can be used to determine which regions of proteins are immunodominant, peptides corresponding to such regions may not always be useful in large-scale screening and diagnosis for example, antigenicity may be lost because the peptide is not in a proper spatial orientation which is recognized by antibodies which react with the protein. Furthermore, as is particularly evident with HIV-I and HIV-2, there is significant genetic variability within each of these two virus groups leading to many serotypes of the viruses. This has put a significant constraint on choosing a region of a protein from which to derive a peptide for use in screening and diagnosis and in formulating vaccines. However, certain immunodominant portions of HIV-1 and HIV-2 proteins have been found to be relatively invariant. It is believed that useful synthetic peptides may be derived from such protein regions.
Recently, such immunologically reactive peptides corresponding to various immunodominant regions of the surface glycoproteins gp120 and gp41 from HIV-1 and the corresponding proteins of HIV-2 encoded by the env gene of the two viruses have been synthesized and shown to react with about 100% efficiency with sera from HIV-1 or HIV-2 infected individuals. When used in assays for detecting the presence of antibodies, such peptides gave no false positive or false negative reactions. See e.g. U.S. patent application Ser. Nos. 051,726 and 051,727 both filed May 18, 1987.
It is believed that a similar approach for diagnosis of HTLV-1 infection using synthetic peptides derived from immunologically important proteins of HTLV-1 would be extremely useful especially in those areas of the world where the virus appears to be endemic.
Several publications have recently presented data showing immunological reactivity of selected synthetic peptides corresponding to antigenic proteins of HTLV-1. In one study several HTLV-1 gag peptides were synthesized. Palker et al., J. Immunology (1986) 136:2393-2397. One of the gag peptides designated SP-71, which corresponds to the C-terminus of the HTLV-1 p19 protein, was found to react with 8/9 HTLV-1 patient sera in a radioimmunoassay (RIA). The amino acid sequence of SP-71 is: Pro-Tyr-Val-Glu-Pro-Thr-Ala-Pro-Gln-Val-Leu. Copeland et al., J. Immunol. (1986) 137:6066-6098, synthesized three additional HTLV-1 peptides which correspond to regions of the protein product encoded by the env gene of HTLV-1. One of these peptides, SP-70, which is located near the C terminus of the major surface glycoprotein gp46, had antigenic activity but reacted with only 4/12 sera from HTLV-1 positive patients. Peptide SP-70 is encoded by the nucleotide sequence of the HTLV-1 genome encompassing base pairs 6066-6098 and has the amino acid sequence: Pro-Pro-Phe-Ser-Leu-Ser-Pro-Val-Pro-Thr-Leu-NH.sub.2.
Synthetic peptides corresponding to regions of immunologically important proteins of HTLV-1 such as gp46 which would react with 100% efficiency with sera from HTLV-1 infected patients would find immediate use in diagnostic methods and as potential immunogenic compositions for eliciting the production of antibodies against HTLV-1.